Analysis of free analyte fractions by rapid affinity chromatography

ABSTRACT

The invention is generally directed toward an analytical method to determine the concentration of the free analyte fraction in a sample. More particularly, the method encompasses applying a sample comprising a free and bound analyte fraction to an affinity column capable of selectively extracting the free fraction in the millisecond time domain. The signal generated by the free fraction is then quantified by standard analytical detection techniques. The concentration of the free fraction may then be determined by comparison of its signal with that of a calibration curve depicting the signal of known concentration of the same analyte.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 09/776,800filed on Feb. 5, 2001, now pending, which is hereby incorporated byreference in its entirety.

This invention was made with Government support under Grant No. 5 R01GM044931 awarded by the National Institute of General Medical Sciences.The Government has certain rights in this invention.

FIELD OF THE INVENTION

The invention is directed toward an analytical method to determine theconcentration of the free analyte fraction in a sample. Moreparticularly, the method encompasses the use of affinity chromatographyto determine the concentration of the free analyte fraction in a sample.

BACKGROUND OF THE INVENTION

Many drugs, hormones, and toxins exist in two distinct forms as theypass through the blood stream: 1) a fraction that is non-covalentlybound to proteins or other blood components and 2) a fraction that isnon-bound, or free, in solution. The free and bound fractions arepresent in a dynamic state, in which solutes in one state arecontinually exchanging with those in the other. Accordingly, inbiological systems this process is constant and an equilibrium is formedbetween the free and bound fractions.

It has long been hypothesized that the free fraction of such substancesis the biologically-active form, since it is this form which crossescell membranes and interacts with cell receptors or other targetligands. Because the free fraction is the biologically-active form, thismakes analysis of free fractions of these substances of particularinterest in clinical chemistry and pharmaceutical science as a means forcontrolling and studying their effects within the body.

For many substances it is possible to use their total concentrations inblood or serum as estimates of their free levels by assuming there is aconstant relationship between these two types of values. However, thereare numerous situations where this approach does not provide meaningful,or even remotely accurate information. For example, after surgery,during malnutrition or pregnancy, and in various disease states (e.g.,cancer, renal failure or liver disease) there can be a large fluctuationin the concentration of binding proteins present in blood. This canshift the equilibrium between these proteins and drugs that bind to themand concomitantly cause a change in a drug's free fraction even thoughits total concentration remains unaffected. Similar shifts indrug-protein binding can occur with age (e.g., in newborns or theelderly) and in situations where several drugs and/or endogenous agentscompete for the same binding proteins. A drug with a high totalconcentration versus its binding proteins also creates problems whentrying to estimate the free fraction based upon total proteinconcentrations since this may result in a non-linear relationshipbetween the drug's total and free levels.

Although several analytical methods have been developed in an attempt todetermine the free fraction, all of these methods are plagued withinherent inaccuracies or are lengthy and tedious to perform. Examples ofthese methods include equilibrium dialysis, ultrafiltration and the useof natural filtrates, such as tears or saliva. A major problem withthese techniques is that the analysis often involves the use of anadditional binding reagent or separation process that interacts with thefree or bound fraction and causes the equilibrium between thesefractions to be altered. For example, techniques with long analysistimes, on the order of several seconds, results in bias in themeasurement process because it allows the release of a significantamount of solutes from the bound fraction which is then detected withthe original free fraction. The end result is an error in the apparentconcentration of free fraction that is measured. In addition, many ofthese techniques suffer from non-specific interactions (e.g., binding ofdrugs to dialysis or filtration membranes), and are limited to onlycertain types of analytes (as is the case with natural filtrates).

Accordingly, a need exists to determine the free fraction withoutimpacting the equilibrium between the free and bound fractions of thesolutes. Equally, a need exists for a method that is highly specific andcan be employed to determine the free fraction of a vast number ofdifferent substances.

SUMMARY OF THE INVENTION

Among the several aspects of the invention therefore, is provided amethod to determine the concentration of a free analyte fraction in asample, the sample comprising a bound analyte fraction and the freeanalyte fraction, the method comprising applying the sample to anaffinity column wherein the column separates the free analyte fractionfrom the sample in the millisecond time domain,detection of signal fromthe free analyte fraction separated from the sample, and determining theconcentration of free analyte present in the sample.

In yet another aspect of the invention is provided a method to determinethe concentration of a free analyte fraction in a sample, the samplecomprising a bound analyte fraction and a free analyte fraction, themethod comprising applying the sample to an affinity column, the columnseparating the sample into the free analyte fraction and the boundanalyte fraction in the millisecond time domain wherein the free analytefraction is adsorbed to the column and the bound analyte fraction passesthrough the column, applying a series of standards to the affinitycolumn wherein each standard applied comprises a known concentration ofthe same free analyte present in the sample, the column separating eachof the standards into a free fraction and a bound fraction in themillisecond time domain wherein the free fraction is adsorbed to thecolumn and the bound fraction passes through the column, detection ofsignal from the free analyte fraction separated from the sample in (a)above, detection of signal from the free analyte fraction separated fromthe standard in (b) above, generating a calibration curve based upon thesignal detected in (d) above, the curve comprising a graph of theconcentration of free analyte present in each standard versus the signaldetected for each concentration, and determining the concentration ofthe free analyte fraction present in the sample by comparing the signaldetected in (c) above with the calibration curve in (e) above.

Yet a further aspect of the invention provides a method to determine theconcentration of a free analyte fraction in a sample, the samplecomprising a bound analyte fraction and a free analyte fraction, themethod comprising applying the sample to an affinity column, the columnseparating the sample into the free analyte fraction and the boundanalyte fraction in the millisecond time domain wherein the free analytefraction is adsorbed to the column and the bound analyte fraction passesthrough the column, applying the sample set forth in (a) to an inertcontrol column wherein the free analyte fraction does not adsorb to thecolumn and a total analyte fraction comprising the free analyte fractionand the bound analyte fraction passes through the column, applying aseries of standards to the affinity column wherein each standard appliedcomprises a known concentration of the same analyte present in thesample, the column separating each of the standards into a free fractionand a bound fraction in the millisecond time domain wherein the freefraction is adsorbed to the column and the bound fraction passes throughthe column, applying the same series of standards set forth in (c) to aninert control column wherein the free analyte fraction does not adsorbto the column and a total analyte fraction comprising the free analytefraction and the bound analyte fraction passes through the column,detection of signal from the bound analyte fraction of the sample in (a)above, detection of signal from the total analyte fraction of the samplein (b) above, detection of signal from the bound fraction of thestandard in (c) above, detection of signal from the total analytefraction of the standard in (d) above, generating a calibration curvebased upon the signal detected in (g) above, the curve comprising agraph of the bound concentration of analyte present in each standardversus the signal detected for each concentration, generating acalibration curve based upon the signal detected in (h) above, the curvecomprising a graph of the total concentration of analyte present in eachstandard versus the signal detected for each concentration, determiningthe bound analyte fraction in the samples by comparing the signal in (e)above with the calibration curve in (i) above, determining the totalanalyte fraction in the samples by comparing the signal in (f) abovewith the calibration curve in (j) above, and determining theconcentration of the free analyte fraction present in the sample bysubtracting the bound analyte fraction in (k) above from the totalanalyte fraction in (1) above.

Still another aspect of the invention provides a method to determine theconcentration of a free analyte fraction in a sample, the samplecomprising a bound analyte fraction and the free analyte fraction, themethod comprising applying the sample to an affinity column wherein thecolumn separates the free analyte fraction from the sample in themillisecond time domain, detection of signal from the free analytefraction separated from the sample and comparing the signal detected in(b) to a standard thereby determining the concentration of free analytepresent in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims and accompanying figures where:

FIG. 1 depicts a drawing of a typical microcolumn that may be employedin the method of the invention.

FIG. 2 depicts change in column void time with column length and solventflow rate for 2 mm ID HPLC columns packed with porous silica. Theseresults assume an overall porosity of 0.80 within the column (i.e., 80%of the column volume is occupied by the mobile phase). Using a columnwith an inner diameter of 1 mm or 4 mm gives similar results but withthe vertical position of-the lines in this graph being lowered or raisedby 4-fold, respectively.

FIG. 3 depicts the reproducibility of stationary phase content in asandwich microcolumn as a function of the number of injections whichwere used to apply a fixed amount of an immobilized hemoglobin supportto a 2.1 mm ID×620 μm microcolumn. These results represent the averageof triplicate analyses.

FIG. 4 depicts release of protein-bound S-warfarin following theinstantaneous removal of this drug's free fraction from an equilibriummixture of S-warfarin and HSA. The graph in (a) shows the progression ofthis reaction over ten seconds, while the plot in (b) shows an expandedview on the sub-second time scale. These graphs were generated for astarting mixture that contained 4.56×10³¹ ⁵ M HSA and 1.1×10⁻⁵ MS-warfarin at 25° C. An equilibrium constant of 3.4×10⁵ M⁻¹ was used todetermine the initial amount of free warfarin in this sample. Afirst-order rate constant of 0.14 s⁻¹ was used to describe thedissociation of S-warfarin from HSA.

FIG. 5 depicts chromatograms for the injection of a 20 μL sample of8×10⁻⁷ M R-warfarin onto (a) a diol-bonded silica column and (b) ananti-warfarin immunoaffinity column at a flow rate of 3.0 mL/min(effective column residence time, 60 ms). Other experimental conditionsare provided in the Materials and Methods of the Example section.

FIG. 6 depicts a schematic of a chromatographic system for theextraction and measurement of free and protein-bound warfarin. Detailson the construction of this system can be found in the Materials andMethods of the Example section.

FIG. 7 depicts a chromatogram for separation of HSA from R-warfarinafter the passage of these compounds as a mixture through ananti-warfarin immunoaffinity microcolumn. The initial sample contained1.1×10⁻⁵ M R-warfarin and 4.5×10−5 M HSA. Other conditions are providedin the text.

FIG. 8 depicts simulated extraction of free warfarin from a mixture ofR-warfarin and HSA. The sample and column conditions used in generatingthis plot were the same as those that were present for the experimentalresults in FIG. 7. The solid bottom line indicates the relative amountof free warfarin that was extracted in the absence of HSA. The solid topline shows the amount of warfarin that was extracted from warfarin/HSAmixtures when the bound warfarin was allowed to dissociate from andrebind to HSA as the sample passed through the column. The dashed lineis shown for reference and represents a case in which the amount ofextracted warfarin was identical to the amount of free warfarin in theinjected samples. These simulations were based on association anddissociation rate constants of 1×10⁵ M⁻¹ S⁻¹ and 0.4 s³¹ ¹ for theinteractions of R-warfarin with HSA at 25° C. The adsorption of warfarinto the immunoaffinity column was described by using a column bindingcapacity of 5×10⁻¹¹ mol and a second-order association rate constant of1×10⁵ M⁻¹ S³¹ ¹.

Abbreviations and Definitions

To facilitate understanding of the invention, a number of terms andabbreviations as used herein are defined below:

“Analyte” or “Target Analyte” are used interchangeably herein, and shallmean the component of the sample that binds to the binding agent presentin the active layer of the microcolumn. The analyte will typicallycomprise the free fraction of a drug, hormone, toxin, metal ion, fattyacid, bilirubin or any other endogenous or exogenous compound.Additionally, the analyte may also comprise any other inorganic ororganic compound capable of being separated from the sample, asdescribed herein.

“Binding Agent”, as utilized herein, shall mean the agent in the activelayer capable of selectively binding the target analyte.

“Binding Compound”, as utilized herein, shall mean the compound that thebound fraction binds. Typically, the binding compound comprises aprotein, cell or any other endogenous or exogenous compound.

“Bound Fraction” or “Bound Analyte Fraction” are used interchangeablyherein, and shall mean the portion of the analyte which is bound to abinding compound.

“Free Fraction” or “Free Analyte Fraction” are used interchangeablyherein, and shall mean the portion of the analyte which is not bound toa binding compound.

“Millisecond Time Domain”, as utilized herein, shall mean any amount oftime less than one second.

“Sandwich microcolumn”, as utilized herein, shall mean an embodiment ofthe invention wherein the microcolumn contains a top inert layer, abottom inert layer and an active layer between the two inert layers.

“Sample” or “Liquid” are used interchangeably herein and shall mean themixture applied to the microcolumn containing the analyte. In additionto the analyte, the sample (liquid) generally also contains a loadingbuffer. Any loading buffer may be employed to the extent that the bufferdoes not interfere with the separation process. The sample may compriseany mixture with an analyte. Typically, however, the sample will becomprised of a biological fluid such as blood, plasma, urine,cerebrospinal fluid, tissue samples, or intracellular fluid.

“Uniform Manner”, as utilized herein, shall mean loading the layers ofthe microcolumn in a manner such that these layers have a substantiallyequal distribution of support in both a horizontal and verticaldirection.

BSA=Bovine Serum Albumin

FPLC=Fast-Protein Liquid Chromatography

HPLC=High-Performance Liquid Chromatography

Description of the Preferred Embodiments

Many compounds, as stated above, exist in two distinct forms as theypass through the blood stream: 1) a fraction that is non-covalentlybound to proteins or other binding compounds and 2) a fraction that isnon-bound, or free, in solution. The free and bound fractions arepresent in a dynamic state, in which solutes in one state arecontinually exchanging with those in the other. Applicants havediscovered a method to accurately determine the free analyte fraction ofa sample comprising a free analyte fraction and a bound analytefraction. The method of the present invention employs the use ofaffinity microcolumns to extract the free fraction of an analyte fromthe sample in the millisecond time domain. The capability of extractingthe free fraction in this time domain allows the free fraction to beseparated without impacting the equilibrium between the free and boundfractions. Applicants' discovery, accordingly, circumvents a majorproblem associated with the long separation times that plague currentdetection techniques. Upon its separation, the concentration of the freefraction is then determined employing standard analytical methods.

I. Affinity Microcolumn Design and Construction

The method of the present invention employs the use of a microcolumn toseparate the free fraction of an analyte from the sample in themillisecond time domain. As utilized herein, the terms “microcolumn” or“column” are used interchangeably and FIG. 1 depicts a typicalmicrocolumn that may be employed in the method of the invention. Asshown in FIG. 1, the microcolumn 1 generally has a tubular configurationwith a first end 2, a second end 3, a passageway 4 there between, and aretaining means at the first 5 and second ends 6 of the microcolumn 1.However, the microcolumn 1 may comprise any number of different shapes,all of which are embodiments of the present invention. The retainingmeans 5,6 typically comprises a mesh or small-pore material that acts tohold the support particles within the column while allowing fluid flowthere through. The microcolumn 1 may also contain end fittings at thefirst 7 and second 8 ends of the microcolumn 1 used to connect thecolumn to the chromatographic system. The microcolumn 1 comprises a thinactive layer 9 to facilitate separation of the analyte from the samplein the millisecond time scale and typically a single inert layer in oneembodiment, to several inert layers in additional embodiments. FIG. 1illustrates an embodiment with a top inert layer 10 and a bottom inertlayer 11.

A salient feature of the current invention is the capability of removinga significant amount of the free fraction of a particular analyte from asample without release of the analyte from its protein-bound fraction.In order to accomplish this task, as delineated above, the extractionprocess is preferably accomplished in the millisecond time domain. Themicrocolumn design described herein is particularly suitable for thisapplication because, due in part to its relatively thin active layer, itcan extract the free fraction within this time range. As utilizedherein, “length” of a layer means the thickness or width of the layer.As illustrated by FIG. 2 (plot derived by calculation), only columnswith active layer lengths in the range of about 100 microns to about 1millimeter allow separation in the millisecond time domain whenemploying standard HPLC flow rates of about 0.1 to about 1.0 mL/min.Accordingly, to achieve separation in this time range, the microcolumnsemployed generally comprise an active layer that may be less than about10 microns in thickness. Typically, however, the active layer is fromabout 10 microns to about 1.1 millimeters in thickness and preferably,is not less than approximately 60 microns in thickness. Applicants havefound that active layers with these dimensions, depending upon thespecific sample applied, are generally capable of extracting an analytein about 1 to about 500 milliseconds. And, more preferably, in less thanabout 200 milliseconds.

In addition to rapid extraction of the free fraction, an additionalsalient feature is the ability of the active layer to bind the targetanalyte with both a high degree of selectivity and with a relativelyhigh binding affinity. The active layer, therefore, typically iscomprised of support particles derivatized with any binding agentpossessing selectivity and having a high binding affinity for the targetanalyte. Preferably, such binding affinity is from about 10² to about10⁶ M⁻¹ or greater. In a preferred embodiment, the binding agents areantibodies raised against the target analyte. The antibodies can beeither monoclonal or polyclonal. However, monoclonal antibodies aregenerally employed in applications where a higher degree of selectivityis desired and polyclonals are more typically utilized in applicationswhere a higher degree of binding 35 affinity is desired. Examples ofother suitable binding agents include nucleic acid ligands (e.g.aptamers), synthetic molecular imprints, antibody fragments (e.g. Fabfragments), antibody related molecules (e.g. chimeras or F_(v), chainfragments), and recombinant proteins that act as antibody mimics. Thebinding agent, once selected, may be isolated in accordance with anygenerally known method.

The binding agent can be derivatized to the support particles by anymethod generally known in the art. However, the method preferablyimmobilizes the binding agent to the support particle in a manner suchthat a relatively high percent of the binding agent is active (i.e.binds the target analyte) after the immobilization process. Suitableimmobilization methods for protein ligands, for example, include theSchiff base method and the carbonyldiimidazole method. The Schiff basemethod is generally employed when immobilizing the binding agent throughfree amine groups.

However, when the binding agent comprises antibodies, applicants havefound that a more preferred approach is immobilization through theantibodies' carbohydrate region because this generally results in anactive layer with a higher number of active binding sites compared towhen immobilization is performed through free amine groups. Any methodknown in the art for immobilization via carbohydrate regions may beemployed.

Additionally, the overall binding capacity of the microcolumn is also animportant feature because it impacts both the time and efficiency ofextraction by the active layer. The binding capacity of the column, inpart, is determined by the number of active binding sites present in theactive layer. Preferably, the minimum number of active binding sites inthe column comprises a ratio of active binding sites to free analyte notless than about 1:1, and even more preferably, not less than about 10:1.However, still more preferably, support particles in the active layerwill be derivatized with the maximum concentration of active bindingagent achievable so that the column has the largest binding capacitypossible.

The active layer, additionally, may comprise a number of differentsupport particles. The support particles, as detailed above, functionprimarily as a surface to immobilize the binding agent. The diameter ofthe particle, however, is an important feature that should be consideredbecause it impacts both the length of the active layer and the amount ofbinding agent that may be immobilized in the active layer (i.e. bindingcapacity of the column). Preferably, the particle size is smaller thanthe length of the desired active layer. Applicants have found that apreferred particle diameter is less than about 10 times to about 20times the length of the active layer because particles within this sizerange facilitate uniform packing of the layer by allowing small packingdefects to average out and produce a more uniform packing cross-sectionfor the support. In addition, the support particles should be able totolerate the flow rates and pressures needed in order to obtain thedesired sample contact time with the active layer. The properties thataffect the pressure and flow rate that may be tolerated by the supportparticles include the diameter of the particle, the particle's shape andthe porosity of the particles. Suitable support particles include porousor nonporous glass, silica and other inorganic supports (e.g., aluminaor zirconia), carbohydrate-based supports (e.g.,beaded agarose), andpolymeric supports (e.g., polymethacryltate or polystyrene basedresins); however, one generally skilled in the art of chromatography canselect other appropriate support particles.

The microcolumn may comprise a single inert layer or several inertlayers, depending upon the application. However, common features sharedby all inert layers, irrespective of their number or position within themicrocolumn, is that they typically should have no substantialinteraction with the target analyte, and should preferably bemechanically stable under the flow rate and pressures employed duringthe separation process. Preferable materials for construction of theinert layer include diol-bonded silica, diol-bonded glass beads, agarosebeads, hydroxylated perfusion media, and glycol coated perfusion media.The various inert layers may be constructed from the same supportparticles or different support particles. However, it is usuallypreferred for the sake of convenience in loading the microcolumn thatthe inert layers comprise the same support particles.

The layers in the microcolumn may comprise either an active layer alone,or an active layer and a single inert layer on top of the active layer(wherein the active layer is in communication with the second endretaining means) such that liquid first passes through the inert layerand then passes through the active layer. The utilization of a singleinert layer in this manner is especially suitable for applications wherethe liquid (containing the sample) is to be applied in only a singledirection to the active layer and the column. The inert layer in thisapplication preferably occupies the entire length of the microcolumnbetween the beginning of the first end of the microcolumn to thebeginning of the active layer so that the entire microcolumn is filledwith support particles. Applicants have found that having the entiremicrocolumn filled with support particles increases both the speed andefficiency of separation. Additionally, the inert layer in thisapplication also preferably acts to distribute the injected sampleevenly across the diameter of the column before the sample reaches theactive layer. This allows for a more uniform application of the sampleto the relatively thin active layer.

The layers in the microcolumn may also comprise an active layersandwiched between a top and a bottom inert layer. As utilized herein,the term “top inert layer” shall mean the layer that liquid first passesthrough prior to reaching the active layer and “bottom inert layer”shall mean the layer where liquid passes after it exits the activelayer. The microcolumn preferably comprises both a top and bottom inertlayer for applications where liquid is to be applied in two directionsto the active layer and the column. At any given time, the flow ofliquid through the column is generally only in a single direction.However, it is sometimes preferable to alternate the flow of liquidthrough the column in order to help wash out any impurities that mayhave built up at the top of the column during the application of liquid.The top inert layer in this application serves the same role asdiscussed above for the application employing a single inert layer e.g.more efficient separation. However, applicants have found that it ispreferable to include the bottom inert layer, even in applications wherefluid flow is in only a single direction, because its inclusionincreases the useful life of the active layer by preventing loss ofsupport particles.

The thickness of the inert layers is not a critical feature and does notaffect the time needed to separate the free fraction of an analyte fromthe sample. In general, as stated above, the top inert layer ispreferably the length that remains between the beginning of the columnand the beginning of the active layer. And, the bottom inert layer, ifit is present, is generally from about 1 to about 5 times the length ofthe active layer. Typically, the top inert layer is thicker than thebottom inert layer.

The choice of a particular type of housing for the microcolumn is alsonot a critical facet. The microcolumn housing, however, preferablyemploys components made of materials that are substantially inert tobiological fluids and in particular, substantially inert to the analyteso as not to interfere with the separation process. Accordingly, anymaterial that is substantially inert may be employed to construct themicrocolumn. Suitable materials include stainless steel, polypropylene,certain plastics and fused silica.

The dimensions of the microcolumn are also not a critical feature. Anysize of microcolumn may be utilized to the extent that the total columnlength is preferably greater than the length of the active layer. Thetotal column length and diameter also preferably allow the use ofsufficiently fast flow rates and pressures to achieve the desiredcontact time between the sample and the active layer. Preferably, themicrocolumn has an internal diameter of about 50 microns to about 2centimeters and a length of about 0.2 millimeters to about 2centimeters. In a particularly preferred embodiment, the microcolumn hasan internal diameter of about 0.5 to about 2.1 millimeters and a lengthof about 1 millimeter to about 2 centimeters.

Applicants have found that thin active layers may preferentially beobtained by loading the support particles comprising the layer into themicrocolumn via a plurality of injections, as described in detail below(e.g. see FIG. 3). The normal method of loading a column, applying thesupport particles in one application to the column with the amount ofsupport particles being in excess of that which is needed to fill thecolumn, is sufficient for standard size chromatography columns becausedue to their size, reliable packing of the support particles may beachieved.

Applicants, however, have found that loading the support particles in asingle injection, for columns with dimensions described herein,generally does not result in an active layer capable of consistentlyachieving separation of an analyte from a sample in the millisecond timescale.

Accordingly, the layers are preferably loaded into the column by aplurality of injections of slurry comprising the support particles. Theslurry may be injected into the column employing any apparatus generallyknown for injecting a slurry into a column, for example, a closed-loopsample application system with either a manual injection valve or anautomatic injection system may be utilized. The slurry, in addition tosupport particles, also preferably comprises a packing solvent orbuffer. The packing solvent employed to load the slurry into the columnis not a critical feature; however, the solvent preferably will not harmthe binding agent present in the active layer. One skilled in the art ofchromatography can readily select both an appropriate apparatus toinject the slurry and appropriate packing solvents.

Applicants have also found, as illustrated by FIG. 3, employing a largernumber of injections and less support per injection, achieves a morecontrolled delivery of support particles because statistical variationsthat occur during the delivery of small amounts of support particles tothe column are averaged out. This is particularly true as layerthickness decreases. Uniform packing of support particles in the layers,especially the active layer, is preferable because it provides morereproducible results for injected samples by allowing parts of thesample that are injected at different locations along the diameter ofthe column to achieve consistent sample contact times with the activelayer. Accordingly, the number of injections to introduce a layer intothe column is generally from about 10 to about 100. More preferably, thenumber of injections to introduce a layer is from about 30 to about 40when the layer length is from about 100 to about 500 microns, and isfrom about 60 to about 80 injections when the layer length is from about60 to about 100 microns in length.

The slurry density (milligrams of support particles per milliliter ofpacking solvent), or amount of support particle applied to the columnper injection, will vary greatly depending upon the desired thickness ofthe layer.

Typically, however, the slurry density will be from about 0.1 to about20 milligrams of support particles per milliliter of packing solvent andmore preferably, will be from about 1 to about 5 milligrams of supportparticles per milliliter of packing solvent. In general, the inertlayer(s) and active layer are loaded at approximately the same slurrydensity. One of ordinary skill in the art can readily determine theappropriate slurry density needed to achieve a layer having a particularthickness when employing a specific number of injections.

The desired slurry density, once selected, is preferably maintainedthroughout column injection in order to facilitate uniform layerpacking. To maintain consistent slurry density, the slurry typicallyundergoes shaking between injections to ensure that the supportparticles are uniformly distributed in the slurry. It is also preferableto monitor the turbidity of the slurry at a wavelength of approximately800 nm to ensure the amount of support particles per milliliter remainsconstant. Furthermore, typically the slurry density is calculated atnumerous points during injection by comparison to slurries of knowndensity employing the same support particles.

Applicants have also found, in addition to loading the support particlesby a plurality of injections, that varying the flow rate and pressureduring column loading also serves to provide a more uniform and thinactive layer. In a particularly preferred application, the pressure andflow rate are increased for a short period of time near the beginningand end of the loading of each layer (as illustrated in Table 1). Thisincreased pressure and flow rate facilitates compression of the layerand distributes the support particles within the layer evenly across thediameter of the column. In a typical column loading procedure, forexample, the flow rate of slurry injection into the column is betweenabout 3 mL/min and about 5 mL/min, with the higher flow rate occurringgenerally at the beginning and end of the loading of each layer.

Additionally, pressure during column loading is typically maintainedbetween about 2000 and about 4000 psi, with a higher pressure preferablyoccurring at the beginning and end of the loading of each layer. Theparticular flow rates and pressures utilized to load each layer of thecolumn is not a critical feature and accordingly, may be variedsignificantly from the general examples provided herein depending uponthe particular application.

Table 1 sets forth a general procedure for loading a 1.0 cmimmunoaffinity microcolumn comprising an active layer between a top andbottom inert layer. The procedure set forth in Table 1 is forillustrative purposes only and shall not be construed to limit the scopeof the present invention as described in greater detail herein.

TABLE 1 General Procedure for Preparing a Microcolumn 1 Assemble columnfittings on the second end of the microcolumn (and retaining means) andattach the microcolumn to the packing apparatus; 2 Make two particlesupport slurries in the packing solvent, one consisting of inert supportparticles, and the other containing the active support particles. For animmunoaffinity microcolumn, the packing solvent employed may be pH 7.0,0.10 M potassium phosphate buffer and the slurry of the inert supportparticles typically may contain a diol-bonded material (e.g. 2 mg/mLdiol-bonded silica). The second slurry contains the immunoaffinitysupport particles at a concentration that is determined by utilizingequation 2 or any other generally known method (as set-forth in theexamples below) and the desired thickness of the final active layer 3Begin flow of the packing solvent through the column. This is generallydone at a rate of approximately 3 mL/min for immunoaffinitymicrocolumns, but is not critical. Make approximately five injections(at 150 uL per injection for a 1.0 cm long column) of the inert supportslurry, followed by an increase in flow rate to approximately 5 mL/minfor approximately 5 minutes 4 Return the flow rate to approximately 3mL/min and make the required number of injections of the active layer(per equation 2 or any other generally known method). After making theseinjections, increase the flow rate to approximately 5 mL/min forapproximately 5 minutes 5 Return the flow rate to approximately 3 mL/minand make enough injections of the inert support slurry to fill theremainder of the column bed 6 After the column bed has been filled,increase the column backpressure to the desired level, typically about3000 to about 4000 psi. Allow the column to equilibrate at this pressurefor approximately 10 minutes. Gradually release the pressure. Remove thecolumn from the packing apparatus and place a frit (retaining means) andend fitting onto the open end of the column. The column is now ready foruse

The microcolumn, in addition to its relatively thin active layer, isalso generally able to tolerate flow rates and pressures during sampleinjection that are capable of achieving the desired sample contact timewith the active layer. The flow rate and pressure depends not only onthe support particles employed in layer construction, but also on thecolumn diameter and upper pressure limit that can be tolerated by thechromatographic system. In general, any flow rate and pressure necessaryto achieve the desired residence time and tolerated by thechromatographic system employed is within the scope of the presentinvention. Typically, however, the microcolumns may be subjected to flowrates of between about 0.01 to about 9.0 mL/min and pressures betweenabout 10 to about 6000 psi. More preferably, the pressure is betweenabout 100 to about 1500 psi.

II. Determination of the Free Analyte Fraction

Encompassed in the method of the invention is a means to determine theconcentration of the free analyte fraction in a ample comprising a freefraction and a bound fraction. The method entails generating acalibration curve comprising data obtained by analyzing a series ofstandards containing a known concentration of the same analyte presentin the sample. The concentration of the free analyte present in thesample, as described in detail below, is then determined by comparisonto the calibration curve.

Applicants have found that accurately determining the concentration offree analyte in a sample preferably involves the rapid and selectiveextraction of this fraction from the sample before significant releasefrom its bound fraction. Additionally, applicants have found that thisrelease generally occurs within a few seconds, particularly when theanalyte binds a serum protein or other binding compound, such as HSA oralpha 1-acid glycoprotein. The affinity microcolumns described in detailabove, therefore, are particularly suited for the method of theinvention because they are capable of selectively extracting the freefraction within the millisecond time domain. The bound fraction, on theother hand, does not bind to the affinity column and is thereforepresent in the liquid fraction that passes through the column.

Accordingly, the method of the invention encompasses applying a sampleto the affinity column (described in detail above) under conditionssufficient to bind the free analyte fraction without significantinterference from its bound fraction, which passes through the columnwithout adsorbing. The method, irrespective of the embodiment, alsoentails applying a series of standards to the same affinity column.“Standard” as utilized herein, shall mean a mixture that contains aknown concentration of the analyte. The standard will preferablycomprise the same analyte as is being detected in the sample anddepending upon the embodiment of the invention, may also comprise abinding compound. “Series of Standards” as utilized herein, shall meanapplying from about two to about five standards with different analyteconcentrations to the column. In another embodiment of the invention,the series of standards may be determined without applying the standardsto the column for each application of the method, such as when thecalibration curve has been determined previously (from analysis of thesame standard in an earlier test) or when the standard comes as a partof a kit. The series of standards will preferably contain concentrationsof free analyte that are substantially comparable to the concentrationof free analyte expected to be present in the sample.

In accordance with the method of the invention, the concentration offree analyte present in each standard is determined. This concentrationmay be determined based upon mass or volume measurements in which aknown concentration of pure analyte is weighed and placed into a knownvolume of solution. Equally, a known volume of solution may be dilutedor combined with another solution to prepare the final standardsolution. In addition to these methods, the concentration may bedetermined by any means generally known in the art.

Additionally, the sample and standard, irrespective of the embodiment ofthe invention, are preferably injected onto the column under conditionsthat optimize a rapid and selective binding of the free analyte to thecolumn. A number of conditions may impact the degree of free analytebinding to the column. These conditions are preferably optimized toachieve a high rate of binding and generally include: 1) an activebinding agent in the column that is 35 capable of binding the freeanalyte, 2) solution conditions (as set forth in more detail below)preferably favorable for binding to occur, 3) the binding capacity ofthe column is preferably equal to or greater than the concentration offree analyte injected into the column (as discussed above), and 4) theresidence time is typically optimized such that the time is preferablylong enough that a significant concentration of the free analyte bindsto the column and yet, short enough in duration to prevent significantdissociation from the bound fraction. The sample and standards may beapplied to the column by any means generally known in the art, such asthrough the use of an injection valve or autoninjector system.

In addition to the parameters set forth above impacting bindingcapacity, a number of operating conditions employed during sample andstandard injection onto the column are also preferably optimized. Onesuch operating parameter is selection of the loading and elutionbuffers. The buffers selected preferably mimic the pH and solventconditions of the sample to ensure that the equilibrium between the freeand bound fractions is not disrupted. For example, when a biologicalsample is being analyzed, any physiological buffer, such as phosphatebuffer, may be employed. And, the buffer typically will have a pH ofapproximately 7.2 to about 7.4 (pH of blood, serum or plasma). Thetemperature during injection of standard/sample onto the column is alsoimportant. Again, the temperature will preferably mimic the naturaltemperature of the sample to avoid disrupting the binding properties ofproteins in the sample prior to their application to the affinitycolumn. Additionally, applicants have found that flow rate during sampleinjection can dramatically impact both extraction efficiency anddissociation of analyte from the bound fraction. Typically higher flowrates result in less dissociation, while slower flow rates increaseextraction efficiency. Therefore, an intermediate flow rate ispreferably employed during column injection and one generally skilled inthe art of chromatography can readily determine this rate, which willvary depending upon the particular application. Typically, however, theflow rate is from about 0.1 to about 10.0 ml/min, and even morepreferably, the flow rate is from about 0.5 to about 1.5 ml/min.

Accordingly, in one embodiment of the invention the concentration offree analyte present in the sample is determined by analyzing data froma series of standards comprising known concentrations of the same freeanalyte present in the sample without the presence of any bindingcompounds. In this embodiment, therefore, the concentration of the freefraction is determined by directly analyzing data from the free fractionof the series of standards (“Direct Method”). The sample, as detailedabove, is applied to the column employing the same operating parametersas with injection of standard onto the column. The column separates thesample and series of standards into a free fraction and a bound fractionin the millisecond time domain. As detailed above, the free fraction ofboth the standard and sample is adsorbed to the column, while the boundfraction passes through the column.

The free analyte fraction of both the standard and sample isolated bythe column is typically then detected as a signal by any means generallyknown in the art of analytical chemistry. “Signal” as utilized herein,shall mean the chemical or physical response that allows the analyte tobe detected (in either the standard or sample). The signal can either begenerated by the analyte itself (e.g. see detection of warfarin in theExample below) or generated by another compound that is linked to theanalyte (e.g. the use of a labeled analog of an analyte to allow theunlabeled analyte to be detected). In addition, the signal is specificto the particular detection method employed. The choice of a particulardetection method is not critical. However, the detection method employedis preferably the same for both the standard and sample in order togenerate a reliable calibration curve. Detection may be performed byeither an on-line or off-line method. An “on-line” method, as utilizedherein, shall mean a method in which there is a direct coupling betweenisolation of the free fraction (via affinity chromatography) and itsdetection such that the isolated fraction is automatically transferredto the detection mechanism through an interface that connects the twosystems. An “off-line” method, on the other-hand, as utilized herein,shall mean a method in which the isolated fraction is collected and thenmanually transferred to the detection mechanism. Suitable detectionmethods include immunoassay, mass spectrometry, gas chromatography, anddetection based upon ultraviolet absorbance, fluorescence detectors, andelectrochemical detectors. Preferably, however, the detection techniqueutilized will comprise an on-line method with direct detection in orderto facilitate efficiency of such detection.

The calibration curve can then be generated after the isolation andsubsequent detection of signal, as detailed above, of the standard withknown concentrations of free analyte. The calibration curve comprises agraph depicting the concentration of free analyte present in eachstandard versus the signal detected for each concentration.

Additionally, the plot can be generated either manually, with aspreadsheet (e.g. Lotus or Excel) or by employing any computer programgenerally known in the art for linear or non-linear regression.

The concentration of the free fraction present in the sample can then bereadily determined utilizing the calibration curve delineated above bysimply comparing the signal detected from the free analyte fractionseparated from the sample with the array of signals depicted on thecalibration curve. This direct comparison is possible because the curvedepicts the signal for known concentrations of the free fraction(generated from the series of standards). Therefore, the concentrationof the free fraction of the sample may be determined by comparing itssignal to signal depicted in the calibration curve for the standardswith known free analyte concentrations.

In yet another embodiment, the concentration of the free analytefraction of the sample is determined by analyzing data from a series ofstandards comprising known concentrations of the same free analytepresent in the sample and a binding compound. In this embodiment, incontrast to the Direct Method detailed above, the concentration of thefree fraction of the sample is determined by analyzing data from thebound fraction and a total fraction (described below) from the series ofstandards (“Indirect Method”). In this embodiment, similar to theembodiment delineated above, the sample and series of standards areapplied to the affinity column employing the same operating parameters.The column separates the sample and series of standards into a freefraction and a bound fraction in the millisecond time domain.Additionally, the free fraction of both the standard and sample isadsorbed to the column while the bound fraction passes through thecolumn. In the Indirect Method, in contrast to the Direct method, thebound fraction of both the sample and series of standards is retainedfor further analysis in the next step of the method.

However, also in contrast to the Direct Method, the Indirect methodemploys the use of an additional inert control column. In thisembodiment, the same sample and series of standards applied to theaffinity column described above are applied to an inert control column.The inert control column comprises a column constructed in all detailslike the affinity column discussed above except that the supportparticles in its active layer are not derivatized with a binding agent.For example, the inert control column and affinity column employed inthis embodiment are the same size, are constructed from the samematerials, and are operated under the same parameters (i.e. pressure andflow rate). However, because the inert control column is not derivatizedwith binding agent, the free analyte fraction is not separated fromeither the sample or series of standards. Instead, a total analytefraction comprising the bound fraction and free fraction pass throughthe column. The total fraction is retained from both the sample andseries of standards for further analysis in the next step of the method.

The signal of the bound fraction of both the sample and series ofstandards is then detected by any means generally known in the art ofanalytical chemistry, as described in detail above for the DirectMethod. In addition, the signal of the total fraction of both the sampleand series of standards is also detected in accordance with theprocedures described above for the Direct Method.

In contrast to the Direct Method, the Indirect Method entails generatingtwo calibration curves. The first calibration curve comprises a graphdepicting the concentration of analyte present in the bound fraction foreach standard versus the signal detected for each concentration. Thesecond calibration curve comprises a graph depicting the concentrationof analyte present in the total fraction for each standard versus thesignal detected for each concentration. As described above, thecalibration curves can be generated either manually, with a spreadsheet(e.g. Lotus or Excel) or by employing any computer program generallyknown in the art for linear or non-linear regression.

In accordance with the method of the invention, the concentration of thefree analyte fraction present in the sample can then be determinedutilizing the two calibration curves described above. The concentrationof the bound analyte fraction present in the sample can readily bedetermined by comparing the signal detected for the bound fraction ofthe sample with the calibration curve depicting the signal detected fromthe bound fraction of the series of standards with known analyteconcentrations. The concentration of the total fraction present in thesample can also be readily determined by comparing the signal detectedfor the total fraction present in the sample with the calibration curvedepicting the signal detected from the total fraction of the series ofstandards with known analyte concentrations. Finally, per this method,the concentration of free analyte present in the sample can bedetermined by simply subtracting the concentration of the bound fractionpresent in the sample from the concentration of the total fractionpresent in the sample.

The methods of the present invention may be employed to determine thefree analyte fraction of any sample comprising a bound and freefraction, to the extent that the free fraction is capable of beingseparated from the sample, as described herein. A typical applicationfor the method, however, is the clinical analysis of the free fractionof a hormone, drug and other endogenous or exogenous agents present inbiological samples. The method may also be employed for thepharmaceutical analysis of free drug levels or drug metabolite levels inbiological samples. Equally, the method may be employed in toxicologystudies to quantify the free fraction of organic compounds and/orinorganic compounds present in a sample. In a research setting, themethod may be utilized to study the mechanism of protein bindingprocesses.

The detailed description set-forth above is provided to aid thoseskilled in the art in practicing the present invention. Even so, thisdetailed description should not be construed to unduly limit the presentinvention as modifications and variation in the embodiments discussedherein can be made by those of ordinary skill in the art withoutdeparting from the spirit or scope of the present inventive discovery.

All publications, patents, patent applications and other referencescited in this application are herein incorporated by reference in theirentirety as if each individual publication, patent, patent applicationor other reference were specifically and individually indicated to beincorporated by reference.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

EXAMPLES

The examples illustrate the ability of the method of the presentinvention to detect the free fraction of warfarin from a samplecomprising warfarin and human serum albumin (“HSA”). Warfarin is oneexample of a drug which has significant binding in blood, and is acommon anticoagulant used in humans for the treatment and prevention ofheart attacks and strokes. It exists in two enantiomeric forms, R-(+)-and S-(−)-warfarin, which are given as a racemic mixture. Both formshave pharmacological activity, but S-warfarin is several times morepotent than the R-enantiomer. In the circulation, R- and S-warfarinexist mainly in their bound forms, with most of this binding occurringwith the protein HSA. Because of warfarin's pharmaceutical significance,binding affinity for HSA, and stereochemistry, it is an excellent modelto illustrate the broad applicability of the method of the currentinvention.

Materials and Methods

Reagents. The R- and S-warfarin were donated by Dupont Pharmaceuticals(Wilmington, Del.). The polyclonal anti-warfarin antibodies were fromAccurate Chemical (Westbury, N.Y.). The HPLC-grade Nucleosil Si-500 andSi-1000 silica (7 μm particle size, 500 Å and 1000 Å pore size) wasobtained from Alltech (Deerfield, Ill.). The 7-amino-4-coumarin-3-acetylhydrazide (AMCA-hydrazide) and reagents for the bicinchoninic acid (BCA)protein assay (Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A.K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.;Olson, B. J.; Klenk, D. C. Anal. Biochem. 1985, 150, 76.) were fromPierce (Rockford, Ill.). HSA (Cohn fraction V, 99% pure, fatty acidfree) and rabbit immunoglobulin G (IgG) were purchased from Sigma (St.Louis, Mo.). All other chemicals were reagent-grade or better. Allaqueous solutions were prepared using deionized water from a Nanopurewater system (Barnstead, Dubuque, Iowa).

Apparatus. Samples for the BCA protein assay were analyzed using aShimadzu UV160U absorbance spectrophotometer (Kyoto, Japan).Immunoaffinity columns were packed using a modified N60 injection valvefrom Valco (Houston, Tex.) and a CM3000 HPLC pump from LDC Analytical(Riviera Beach, Fla.). The final chromatographic system used in theanalysis of free and bound warfarin consisted of one anti-warfarinimmunoaffinity microcolumn (prepared as described later) in series withtwo 5 cm×2.1 mm ID Pinkerton GFF II internal surface reversed-phase(ISRP) columns from Regis Technologies (Morton Grove, Ill.). Detectionin the chromatographic system was performed by a Shimadzu RF-535fluorescence monitor. Samples were injected by an AS3000 autosamplerfrom Thermoseparations (Schaumberg, Ill.). The application and elutionbuffers for the immunoaffinity column were delivered using PU-980 HPLCpumps from Jasco (St. Louis, Mo.). A LABPro automated six-port valvefrom Rheodyne (Cotati, Calif.) was used to switch between these buffers.An organic modifier for displacement of the HSA-bound warfarin wasintroduced to the effluent of the immunoaffinity column through the useof a post-column mixing tee and a CM3200 pump from LDC Analytical. Thechromatograms were collected on a 300 MHZ Pentium computer from TCE(Hoffman Estates, Ill.) with Winner-on-Windows software fromThermoseparations. The temperature of the system was controlled using aVWRbrand 13L immersion circulating water bath from VWR Scientific (WestChester, Pa.).

Preparation of Immunoextraction Column. Antibodies were purified usingAMCA-hydrazide as an affinity ligand. AMCA is a coumarin derivative thatis similar in structure to warfarin. It was used here to isolateanti-warfarin antibodies from their corresponding antiserum. TheAMCA-hydrazide was coupled to Nucleosil Si-1000 silica in a mannersimilar to that reported for the synthesis of dihydrazide-activatedsilica (Ruhn, P. F.; Garver, S.; Hage, D. S. J. Chromatogr. A 1994, 669,9.). The anti-warfarin antibodies were isolated with this support byincubating one milliliter of anti-warfarin antiserum with 300 mg of theAMCA silica for two hours at room temperature. After 15 incubation, thesamples were centrifuged and the supernatant was removed. The supportwas then washed with pH 2.5, 0.10 M potassium phosphate buffer for 10min, followed by a second centrifugation step. This second supernatant(containing purified anti-warfarin antibodies) was then collected,adjusted to pH 7.0 with a small concentration of 1.0 M sodium hydroxide,and stored at 4° C. until further use.

Diol-bonded Nucleosil Si-500 silica was prepared as described previously(Ruhn, P. F.; Garver, S.; Hage, D. S.

J. Chromatogr. A 1994, 669, 9.). The diol coverage of this support was127±4 (1 SD) μmol/g of silica, as determined in replicate by aniodometric capillary electrophoresis assay. The purified anti-warfarinantibodies were immobilized to this diol-bonded silica by the Schiffbase method (Larsson, P.-O. Methods Enzymol. 1984, 104, 2121.). Theprotein content of the resulting immunoaffinity support was determinedby a BCA assay to be 2.05±0.05 mg antibodies/g silica (or 14 nmol/g),using rabbit IgG as the standard and diol-bonded silica as the blank.

The anti-warfarin immunoaffinity support was used to pack a sandwichmicrocolumn, as described herein. This column had an inner diameter of2.1 mm and a total length of 1.0 cm, with a 1.1 mm portion containingthe immunoaffinity support and the remainder containing an inert layerof diol-bonded silica. The immunoaffinity layer was placed within thiscolumn by making thirty-two 250 μL injections of a 0.3 mg/mL slurry ofthe anti-warfarin support in pH 7.0, 0.10 M phosphate buffer. Theremainder of the column was filled in a similar manner with diol-bondedNucleosil 500.

Chromatographic Studies. The extraction studies were performed by making20 μL injections of 0.8−5×10⁻⁷ M warfarin at flow rates ranging from 0.5to 3.0 mL/min. Five replicate injections were made at each flow rateusing microcolumns that contained the immunoaffinity support or onlydiol-bonded silica. The non-retained peaks observed with theimmunoaffinity column were compared to those seen on the diol column todetermine the relative concentration of warfarin that had been removedby the immobilized antibodies. The application buffer in these studieswas pH 7.0, 0.10 M potassium phosphate and the elution buffer was pH2.5, 0.10 M potassium phosphate. Between injections, the immunoaffinitycolumn was washed for 5 min with the elution buffer at 1.0 mL/min andwas allowed to regenerate for 10 min in the application buffer at 1.0mL/min.

The separation of HSA and HSA-bound warfarin was studied by injecting 20μL of warfarin/HSA mixtures onto a diol microcolumn that was in serieswith two ISRP columns.

The samples in this study contained 0−3.5×10⁻⁵ M warfarin and 3 mg/mL(4.5×10⁻⁵ M) HSA in pH 7.0, 0.10 M phosphate buffer. These samples wereinjected at 1.0 mL/min in the presence of the pH 7.0 application buffer.A separate solvent stream was introduced directly after the microcolumnusing a three-way mixing tee. In the final optimized system, this secondsolvent stream contained 7.5% 1-propanol in pH 7.0, 0.10 M phosphatebuffer and was added at a flow rate of 0.2 mL/min to induce dissociationof HSA-bound warfarin. The same chromatographic system was used todetermine the concentration of free warfarin in warfarin/HSA mixtures byreplacing the diol column with the immunoaffinity microcolumn. A seriesof twenty replicate injections were made in this experiment usingsamples that contained well-defined concentrations of R- or S-warfarinand HSA. The non-retained peak areas for the warfarin in these sampleswere then compared to the areas obtained for warfarin/HSA standards withthe diol column.

Computer Simulations. The simulations of warfarin extraction anddissociation were performed on an IBM-compatible computer using programswritten in Turbo C++ (Borland International, Scotts Valley, Calif.).This was accomplished by using a grid propagation algorithm that haspreviously been used to examine the adsorption of analytes toimmobilized antibodies and similar ligands in affinity columns. Thisalgorithm was modified to include the reversible binding of an analyteto an agent in the mobile phase. In this method, the column was dividedinto a large number of slices of equal width. As an analyte movedthrough this column, its binding to the immobilized ligand and solubleagent in each slice was described by using mass balance and thedifferential equations for the rates of these reactions. This system ofequations was solved for that particular slice and interval of time byusing a fourth-order Runga-Kutta method (Margenau, H.; Mosely, G. M. TheMathematics of Physics and Chemistry; Van Nostrand; Princeton, 1956.).Flow through the column was simulated after each iteration by taking thecompounds that remained in the mobile phase and moving these onto thenext slice. The process of distributing and moving the analytes wasrepeated until all of the analyte had either bound to the immobilizedligand or had left the column. The relative concentration of analytethat had adsorbed to the column was then calculated, thus providing theretained fraction. Convergence of these results was tested by performinga series of related simulations in which the column was divided into anincreasing number of slices but with a decreasing concentration of timebeing used per iteration in each slice. This gave a maximum estimatederror of less than 0.2% in the calculated concentration of retainedanalyte.

EXAMPLE 1 Initial Selection of Conditions for Free Drug Extractions

Previous reports have measured the equilibrium and rate constants forthe binding and dissociation of R- and S-warfarin with HSA under avariety of conditions (Loun, B.; Hage, D. S. Anal. Chem. 1994, 66, 3814and Loun, B.; Hage, D. S. Anal. Chem. 1996, 68, 1218.). Based on thisinformation, it was possible to estimate the time needed to remove thefree fraction of warfarin from a warfarin/HSA mixture without havingthis fraction contain a significant concentration of warfarin that hadbeen released from its protein-bound form. Some plots that were used tostudy the extent of this dissociation process are shown in FIG. 4 Theseparticular results were calculated for S-warfarin at 25° C. underconditions similar to those used in this study.

This type of plot was also generated for R-warfarin, which has aslightly faster rate of dissociation from HSA under the givenexperimental conditions.

The model used in FIG. 4 assumes 1) that there is an instantaneousremoval of all free warfarin from the initial sample, 2) that anywarfarin which is later released from its complex with HSA will bindimmediately to the extraction column, and 3) that the concentration ofextracted warfarin is much smaller than the binding capacity of thecolumn. Based on this model, it was found that roughly 75% of theS-warfarin that was originally bound to HSA would be released from thisprotein and adsorbed to the extraction column within 10 s. Thisindicated that ordinary immunoaffinity extractions, which often takeseveral minutes to perform, (Hage, D. S. J. Chromatogr. B 1998, 715, 3.)would be much too long for separating the free and bound fractions ofS-warfarin. An expanded view of this dissociation process, illustratedin FIG. 4 (b), indicated that extraction times of only a few hundredmilliseconds would be required to isolate the free fraction ofS-warfarin while avoiding any appreciable contamination by warfarin thathad been later released from HSA. The same conclusion was reached incalculations that were performed for R-warfarin.

Although it was useful in FIG. 4 to assume that there was an excess ofbinding sites in the column and that there was immediate removal of anynon-complexed warfarin from solution, this does represent a worst-casescenario and probably resulted in estimates for the usable extractiontimes which were smaller than those that might be possible in practice.For instance, either the presence of a finite number of binding sites inthe column or a finite rate of adsorption for the non-complexed warfarinwould reduce any perturbation of the HSA-bound fraction of warfarin asthis passes through the immunoaffinity column. The same thing wouldhappen if the warfarin in solution were allowed to reassociate with HSAinstead of being removed by the column. The effects of eliminating theseassumptions will be considered through the use of computer simulations.However, the model used in FIG. 4 was still found to be a good startingpoint for estimating the conditions needed in an immunoaffinity columnfor isolating a drug's free fraction.

Design of Immunoaffinity Column. Based on FIG. 4, an extraction time ofless than 200 ms was set as the initial goal for the removal of freewarfarin from warfarin/HSA mixtures at room temperature. Under theseconditions, an error of less than 20-40% in the measured free fractionsof R- and S-warfarin was expected due to the dissociation of theirprotein-bound fractions. In order to work within this time frame it wasnecessary to prepare a column that was 35 capable of operating in themillisecond time domain. This was accomplished by using a sandwichmicrocolumn that was prepared as described herein. This consisted of a2.1 mm ID ×1.0 cm tube that was packed with a 1.1 mm thick layer of asupport that contained immobilized anti-warfarin antibodies. Theremainder of the column was filled with diol-bonded silica. The purposeof the immunoaffinity support was to extract warfarin from samples,while the diol-bonded silica was used to hold the immunoaffinity supportin place and to provide uniform sample application to this layer.

In this type of column the actual time over which free drug extractionand sample perturbation takes place is represented by the time duringwhich any given part of the sample passes through the immunaffinitylayer. For instance, an effective extraction time of 200 ms or lesswould be obtained by using a flow rate of at least 0.9 mL/min on a 2.1mm ID column that contains a 1.1 mm layer of an immunoaffinity support.This is well within the range of usable conditions for these columns,which have been operated at flow rates as high as 9-10 mL/min.

The binding capacity of the immunoaffinity columns also had to beconsidered to ensure that overloading did not occur when warfarinsamples were injected onto these columns. The concentration of warfarinbinding sites in one anti-warfarin microcolumn was estimated to be 50pmol. Although this is a relatively small concentration of ligand, it isstill three-to-five times larger than the concentration of free warfarinthat was applied in any sample, thus indicating that these columns didhave a sufficient binding capacity for this present study. This wasconfirmed experimentally by measuring the maximum concentration ofwarfarin that could bind to the microcolumn as the concentration ofinjected warfarin was varied.

One question that remained was whether this type of microcolumn could beused to extract warfarin on the millisecond time scale. This was testedby injecting samples that contained only R- or S-warfarin at variousflow rates. The results obtained on the immunoaffinity column were thencompared to those observed for the same samples and flow rates on aninert control column. FIG. 5 shows an example of such a study. It wasfound that in 120 ms there was 95% extraction of a 5×10⁻⁷ M warfarinsample (i.e., a concentration corresponding to the free warfarin contentexpected for the warfarin/HSA mixtures examined later in this report).At even lower concentrations, such as the 8×10⁻⁸ M warfarin sample shownin FIG. 5, essentially complete extraction was possible in 60 ms. Thus,it was concluded that immunoaffinity microcolumns could be used toextract warfarin on the same time scale that was needed for separatingwarfarin's free and protein-bound fractions.

EXAMPLE 2 Separation of Free and Bound Warfarin Fractions.

The next section of this study began to examine the use of animmunoaffinity microcolumn to measure the free drug fractions ofmixtures that contained known concentrations of HSA and R- orS-warfarin. This was to be done by monitoring the fluorescence of anywarfarin that passed non-retained through the immunoaffinity column. Onedifficulty with this approach is that warfarin has a significant changein its degree of fluorescence when it is in solution versus bound toHSA. This, plus the inherent fluorescence of HSA, created a problem indetection because the non-retained samples were expected to contain amixture of HSA, HSA-bound warfarin, and warfarin that had dissociatedfrom HSA.

The approach used to overcome this problem was to pass the non-retainedsample peaks through a series of ISRP (internal surface reversed-phase)columns. This was done by using the system illustrated in FIG. 6.Although all of the bound warfarin would eventually be released from HSAin such a system, a dissociating agent (1-propanol) was added to themicrocolumn effluent to increase the rate of this process.

The 1-propanol also acted as an organic modifier to aid in the elutionof components on the ISRP columns.

A typical chromatogram for this system is shown in FIG. 7. Asdemonstrated in earlier studies, proteins like HSA are too large to fitin the pores of an ISRP column and will elute in its excluded volume.However, smaller molecules like warfarin can enter these pores andinteract with the hydrophobic phase that is located there. This willcause these molecules to be retained and to elute after proteins fromthese columns. In the case of this study, the second peak was muchbroader than the first since it was produced by warfarin that enteredthe pores at different points along the column as it began to undergodissociation from HSA.

ISRP columns have been used alone for the analysis of free and boundwarfarin in the presence of bovine serum albumin. In these earlierstudies, the free warfarin was retained at the beginning of the ISRPcolumn, while the bound fraction underwent dissociation from albumin andwas retained further downstream. It was shown that this resulted inthree overlapping peaks: the first representing the non-retainedprotein, the second corresponding to warfarin which dissociated fromthis protein, and the third (and slowest eluting peak) representingwarfarin's initial free fraction. It is important to note in thispresent study that the free warfarin peak was not observed on the ISRPcolumn when the samples had previously passed through the immunoaffinitycolumn. This occurred because the free fraction had now been removedfrom these samples. This allowed a much cleaner separation to beobtained between the bound and free fractions of this drug and itsbinding proteins than was reported when using only ISRP supports.

Various factors were adjusted to obtain the separation shown in FIG. 7.The rate of warfarin dissociation from HSA was increased in the ISRPcolumn by adding 1-propanol to the effluent of the immunoaffinitycolumn. This resulted in a sharper peak for the bound warfarin fractionsince it was now released over a shorter distance within the ISRPcolumn.

A 7.5% solution of 1-propanol was found to be optimum for this purposesince it allowed complete dissociation of warfarin from HSA in areasonable time while still providing a large enough difference inretention for their separation. A longer length for the ISRP support wasalso employed (going from one 5 cm long column to two 5 cm columns) toincrease the efficiency of this system and its ability to resolve theHSA and bound warfarin peaks. The result was a separation in whichbaseline resolution was obtained within 5 min of sample injection.

One small problem in using multiple ISRP columns was that this increasedthe back pressure of the overall chromatographic system. This placed alimit on the maximum flow rate and minimum sample residence time thatcould be used with the immunoaffinity microcolumn. As a compromisebetween extraction time, sample throughput and these pressurelimitations, an injection flow rate of 1.0 mL/min was chosen for thefinal system. This gave a residence time of 180 ms for samples in theimmunoaffinity column. As noted earlier, this concentration of time notonly was sufficient to allow the quantitative extraction of warfarin,but it also fell within the range of 200 ms or less that was initiallyselected for use in separating warfarin's free and bound fractions.

EXAMPLE 3 Analysis of Free Warfarin Fractions

The fourth phase of this study used the immunoaffinity and ISRP systemto measure the concentration of free warfarin in known mixtures ofwarfarin and HSA. To quantitate the free warfarin in these samples,standard curves were first prepared in which various concentrations ofR- or S-warfarin were mixed with HSA. These mixtures were then injectedonto the system shown in FIG. 6, with diol-bonded silica being used inplace of the immunoaffinity support in the extraction column. The areaof the warfarin that eluted from the ISRP columns (now corresponding toboth the bound and free warfarin fractions) was measured and plotted asa function of the total warfarin concentration in the sample. Both R-and S-warfarin gave linear responses in these plots with correlationcoefficients of 0.9991-0.9993 for ten samples that contained warfarinconcentrations of 0−1.7×10⁻⁵ M. The lower limit of detection of thismethod for these enantiomers was about 0.5−1×10⁻⁸ M (S/N=2). Thisdetection limit was 50 to 100-fold smaller than the free warfarinconcentrations which were measured in this study.

The warfarin and HSA mixtures that were being used as test samples werenow injected onto the same system in the presence of 1) a microcolumnthat contained only diol-bonded silica or 2) the immunoaffinity columnthat had previously been developed for the millisecond-scale extractionof warfarin. The peak area obtained with the diol column allowed thetotal concentration of warfarin in the sample to be determined. The areameasured after extraction by the immunoaffinity column allowed theconcentration of bound warfarin to be estimated. By combining these twovalues it was then possible to calculate the concentration of freewarfarin in the sample.

This approach was tested by injecting a set of samples that contained0.95−1.10×10⁻⁵ M R- or S-warfarin and 4.5×10⁻⁵ M HSA. Both the R- andS-warfarin samples were injected twenty times onto the diol column andimmunoaffinity column. This gave relative standard deviations of ±4-6%for their measured peak areas on the ISRP system. The free fractionsthat were obtained for these samples are shown in Table 2, below. ForR-warfarin, the free fraction was determined to be 11.8±0.6% at 25° C.(or a bound fraction of 88.2%), while for S-warfarin the free fractionwas 5.9±0.2% (or 94.1% bound).

TABLE 2 Measured and predicted free fractions for R- and S-warfarin insamples containing known mixtures of warfarin and HSA^(a) Measured FreePredicted free Compound Fraction Fraction^(b) R-Warfarin 11.8 (±0.6) %10 (±2) % S-Warfarin  5.9 (±0.2) %  7 (±1) % ^(a)All values shown inparentheses represent a range of ±1 SD. ^(b)The predicted free fractionswere determined by using the known compositions of the samples andequilibrium constants which have previously been measured for thebinding of warfarin enantiomers to HSA at 25° C. The equilibriumconstants that were used for R- and S-warfarin were 2.6 (±0.1) × 10⁵ M⁻¹and 3.4 (±0.1) × 10⁵ M⁻¹, respectively.

The accuracy of these free fractions was evaluated by comparing themwith the predicted results for these samples. This was accomplished byusing the known composition of these samples and equilibrium constantsthat have previously been determined for the binding of R- andS-warfarin to HSA under conditions similar to those used in this study(Loun, B.; Hage, D. S. Anal. Chem. 1994, 66, 3814.). It was assumed inthese calculations that essentially all of the HSA was active; this wasconfirmed experimentally through the use of fluorescence and asolution-phase titration of the HSA with increasing concentrations ofwarfarin. For R-warfarin, it was predicted that approximately 10±2% ofthis drug would be in the free form at equilibrium for the given samplecomposition, while for S-warfarin a predicted free fraction of 7±1 wasobtained. Both predicted results were within one standard deviation ofthe experimental free fractions, thus indicating that there was goodagreement between these values.

An inherent assumption made throughout this project was that theanti-warfarin antibodies in the immunoaffinity column would be able todistinguish between the free warfarin in solution and its HSA-boundfraction. For this to be true, these antibodies would have to interactwith regions in the structure of warfarin that were not exposed whenthis drug was complexed with HSA. The agreement between the actual andpredicted results in Table 2 indicates that this was indeed the case.This specificity for the free versus bound fractions was not surprisingin the case of R-warfarin, which is believed to interact deep within itsbinding site on HSA. But a similar specificity was noted for S-warfarin,which is thought to interact more with HSA's outer surface. This resultindicates that it should be possible to use immunoaffinitychromatography to isolate the free fractions of drugs that have avariety of different orientations in their drug-protein complexes.However, as a precaution, it is still recommended that antibodyspecificity always be considered for any new compounds that are to beexamined by this analytical technique.

EXAMPLE 4 Simulation of Warfarin Extraction and Dissociation

After the free fractions of R- and S-warfarin had been determined intest samples, a comparison was made between the accuracy of these valuesand the errors that had been anticipated due to the dissociation ofbound warfarin during such measurements. For instance, the differencesin the measured and predicted free fractions in Table 2 (+18% forR-warfarin and −16% for S-warfarin) were much smaller than the errors of20-40% that were expected from FIG. 4 and related plots at comparableextraction times. It was suspected from this that these earlier graphshad overestimated the role played by dissociation effects during theisolation of free drug fractions by immunoaffinity chromatography.

A more complete picture of the immunoaffinity extraction process wasobtained through the use of computer simulations. This was accomplishedby developing a model which no longer made the same assumptions thatwere used in FIG. 4. For instance, this model allowed the free anddissociated warfarin to undergo a continuous (rather than instantaneous)extraction on the immunoaffinity microcolumn. It was also now possibleto consider the use of a column with a finite binding capacity andsamples in which any non-complexed warfarin could bind to HSA instead ofthe immobilized antibodies. This was accomplished by adapting a previousalgorithm that has been used to study the adsorption of analytes toaffinity supports, (Hage, D. S.; Walters, R. R. J. Chromatogr. 1988,436, 111; Rollag, J. G.; Hage, D. S. J. Chromatogr. A 1998, 795, 185;and Hage, D. S.; Thomas, D. H.; Roy Chowdhuri, A.; Clarke, W. Anal.Chem. 1999, 71, 2965.) with the inclusion of a reversible solution-phasereaction between the injected drug and its binding proteins. Furtherdetails on this approach can be found in the Methods and Materials.

FIG. 8 shows the simulation results that were obtained for R-warfarin.Similar plots were generated for S-warfarin. The sample and columnconditions that were used in these simulations were the same as thosethat were present in the experimental determination of the free warfarinfractions. The rate constant for adsorption of warfarin to theimmobilized antibodies was estimated from the earlier studies thatexamined the extraction of R- and S-warfarin by the immunoaffinitycolumn.

The lower plot in FIG. 8 shows how the extraction efficiency of warfarinwas predicted to change as this drug was injected at various flow rates.This extraction had its highest efficiency at low flow rates and adecreasing efficiency at higher flow rates. This occurs because there isa smaller contact time between the sample and the immobilized antibodiesas higher application flow rates are used.

The top plot in FIG. 8 shows how the relative concentration of extractedwarfarin compared to the true free fraction in warfarin/HSA mixtureswhen the bound warfarin in these samples was allowed to undergodissociation and rebinding to the HSA. As expected, higher flow ratesand shorter column residence times resulted in less dissociationeffects. This, in turn, allowed the concentration of extracted warfarin(free plus dissociated warfarin) to approach the concentration of freewarfarin that was actually removed from the sample.

One observation that can be made by comparing the two plots in FIG. 8 isthat there will be an optimum flow rate range over which the bestestimates can be made of a drug's true free fraction. For instance, FIG.8 shows that the extent of warfarin dissociation from HSA will beminimized by operating at high flow rates; however, this decreasesextraction of the free warfarin fraction. Working at slower flow ratesprovides a better extraction efficiency but suffers from greater errorsdue to bound drug dissociation. It is only at intermediate flow ratesthat a balance is obtained between accuracy and extraction recovery. Inthis particular case, it was predicted that a flow rate range of roughly0.9-1.6 mL/min was needed to obtain an error of ±15% or less in themeasured free fractions. This compares well with the approximate errorsof +18% and −16% that were estimated for R- and S-warfarin under theseflow rate conditions.

Another observation that can be made from FIG. 8 is that the freefraction errors predicted by the simulations were less than those thatwere originally estimated through the use of simple drug-proteindissociation. For instance, it was determined from FIG. 4 thatdissociation of S-warfarin from HSA would increase the measured freefraction of this drug by 25-30% at an extraction time of 180 ms.However, the more complete model used in the simulations indicated thata maximum error of only 15% would be expected.

In light of the detailed description of the invention and the examplespresented above, it can be appreciated that the several aspects of theinvention are achieved.

It is to be understood that the present invention has been described indetail by way of illustration and example in order to acquaint othersskilled in the art with the invention, its principles, and its practicalapplication. Particular formulations and processes of the presentinvention are not limited to the descriptions of the specificembodiments presented, but rather the descriptions and examples shouldbe viewed in terms of the claims that follow and their equivalents.While some of the examples and descriptions above include someconclusions about the way the invention may function, the inventor doesnot intend to be bound by those conclusions and functions, but puts themforth only as possible explanations.

It is to be further understood that the specific embodiments of thepresent invention as set forth are not intended as being exhaustive orlimiting of the invention, and that many alternatives, modifications,and variations will be apparent to those of ordinary skill in the art inlight of the foregoing examples and detailed description. Accordingly,this invention is intended to embrace all such alternatives,modifications, and variations that fall within the spirit and scope ofthe following claims.

What is claimed is:
 1. A method to determine the concentration of a freeanalyte fraction in a sample, the sample comprising a biological fluidhaving a bound analyte fraction and the free analyte fraction, themethod comprising: (a) applying, the sample to an affinity column havingan active layer which selectively binds free analyte relative to boundanalyte wherein the active layer separates the free analyte fractionfrom the bound analyte fraction of the sample in the millisecond timedomain; (b) detection of signal from the free analyte fraction separatedfrom the sample; and (c) determining the concentration of free analytepresent in the sample.
 2. The method of claim 1 wherein the affinitycolumn comprises an active layer, the active layer comprising supportparticles derivatized with a binding agent.
 3. The method of claim 2wherein the active layer is from about 10 microns to about 1.1millimeters in length.
 4. The method of claim 2 wherein the active layeris not less than 60 microns in length.
 5. The method of claim 2 whereinthe binding agent is selected from the group of agents consisting ofantibodies, aptamers, antibody fragments, synthetic molecular imprints,antibody related molecules and recombinant proteins.
 6. The method ofclaim 5 wherein the binding agent is antibodies.
 7. The method of claim6 wherein the binding agent has a high binding affinity for the freeanalyte fraction.
 8. The method of claim 7 wherein the binding agent hasa binding affinity for the free analyte fraction from about 10² to about10⁶ M⁻¹.
 9. The method of claim 7 wherein the binding agent has abinding affinity for the free analyte fraction greater than about 10⁶M⁻¹.
 10. The method of claim 7 wherein the free analyte fraction isseparated from the sample from about 1 to about 500 milliseconds afterinjection of the sample into the microcolumn.
 11. The method of claim 10wherein the free analyte fraction is separated from the sample fromabout 1 to about 100 milliseconds after injection of the sample into themicrocolumn.
 12. The method of claim 10 wherein the signal is detectedfrom the free analyte fraction by an off-line method or an on-linemethod.
 13. The method of claim 12 wherein the signal is detected by anon-line method with direct detection.
 14. The method of claim 12 whereinthe signal is detected by a method selected from the group consisting ofimmunoassay, mass spectrometry, gas chromatography, ultravioletabsorbance, fluorescence detectors, and electrochemical detectors.
 15. Amethod to determine the concentration of a free analyte fraction in asample, the sample comprising a bound analyte fraction and the freeanalyte fraction, the method comprising: (a) applying the sample to anaffinity column wherein the column separates the free analyte fractionfrom the sample in the millisecond time domain; (b) detection of signalfrom the free analyte fraction separated from the sample; and (c)comparing the signal detected in (b) to a standard thereby determiningthe concentration of free analyte present in the sample.
 16. The methodof claim 12 wherein the active layer is introduced by a plurality ofinjections.
 17. The method of claim 16 wherein the active layer isintroduced by about 10 to about 100 injections.
 18. The method of claim17 wherein the active layer is introduced by about 30 to about 40injections when the active layer is from about 100 to about 500 micronsin length.
 19. The method of claim 17 wherein the active layer isintroduced by about 60 to about 80 injections when the active layer isfrom about 60 to about 100 microns in length.
 20. The method of claim 17wherein the support particles comprising the active layer are injectedonto the column at a density of from about 0.1 to about 20 milligrams ofsupport particle per milliliter of packing solvent.
 21. The method ofclaim 16 wherein the biological fluid is selected from the groupconsisting of blood, plasma, urine, cerebrospinal fluid, a tissuesample, and intracellular fluid.
 22. The method of claim 21 wherein thebinding capacity of the active layer comprises a ratio of the number ofactive binding sites to amount of free analyte present in the samplebetween about 1:1 to about 10:1.
 23. The method of claim 22 wherein thesample is injected onto the column at a flow rate of about 0.01 to about9.0 milliliters per minute.
 24. The method of claim 23 wherein thesample is injected onto the column at a pressure of about 100 to about1500 psi.